The biology of the secretory pathway is of central importance to physiological insulin production: I) Pancreatic beta-cells normally synthesize and export massive quantities of proinsulin, and these quantities are further increased upon elevation of blood glucose. This poses a challenge to the secretory pathway protein folding system and exposes the cells to risk of proteotoxicity. One mechanism that limits the translation of islet proinsulin, especially after elevation of extracellular glucose, involves signaling at the endoplasmic reticulum (ER) by the PERK transmembrane protein kinase that phosphorylates eukaryotic translational initiation factor eIF2alpha on Ser51, which diminishes translation of secretory proteins while allowing the continued translation of a subset of mRNAs including those for ER chaperones. Deficient signaling via this pathway results in a failure to normally downregulate secretory protein translation, and PERK-/- mice exhibit phenotypes that include apoptotic beta-cell failure. Similarly, expression of the Akita [Cys(A7)Tyr] mutant proinsulin that has improper disulfide bonding reportedly also induces apoptosis of beta-cells, even in the absence of genetic defects in ER signaling. In Aim #1, we propose to pursue the hypothesis of disulfide mispairing for a fraction of wild-type proinsulin, and the possibility that this misfolding can lead to beta-cell toxicity - even more so in cells rendered susceptible because of insufficient ER stress response. In Aim #2, we wish to pursue established and new models of misfolded, disulfide-mispaired mutant proinsulin, as well as the possibility of molecular/genetic rescue of cells from death due to proteotoxicity. II) It is generally believed that the fidelity of sorting-packaging functions within the TGN and immature secretory granules is supported by active recycling of membrane proteins between the biosynthetic and endocytic pathways. In Aim #3, we now propose to extend a two-pronged approach for studies in beta-cells involving molecular perturbation of candidate gene products relevant to protein egress from the biosynthetic pathway, as well as endosomal return, in order to examine the relationship of these steps to insulin secretory granule biogenesis.